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Published as doi: 10.1096/fj.07-9458com.
 (The FASEB Journal. 2008;22:1246-1257.)
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(The FASEB Journal. 2008;22:1246-1257.)
© 2008 FASEB

Role of the acidic N' region of cardiac troponin I in regulating myocardial function

Sakthivel Sadayappan*, Natosha Finley{dagger}, Jack W. Howarth{dagger}, Hanna Osinska*, Raisa Klevitsky*, John N. Lorenz{ddagger}, Paul R. Rosevear{dagger} and Jeffrey Robbins*,1

* Division of Molecular Cardiovascular Biology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA;

{dagger} Molecular Genetics, Biochemistry and Microbiology; and

{ddagger} Molecular and Cellular Physiology, Department of Medicine, University of Cincinnati, Cincinnati, Ohio, USA

1Correspondence: Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, MLC 7020, 3333 Burnet Ave., Cincinnati, OH 45229-3039, USA. E-mail: jeff.robbins{at}cchmc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiac troponin I (cTnI) phosphorylation modulates myocardial contractility and relaxation during β-adrenergic stimulation. cTnI differs from the skeletal isoform in that it has a cardiac specific N' extension of 32 residues (N' extension). The role of the acidic N' region in modulating cardiac contractility has not been fully defined. To test the hypothesis that the acidic N' region of cTnI helps regulate myocardial function, we generated cardiac-specific transgenic mice in which residues 2–11 (cTnI{Delta}2–11) were deleted. The hearts displayed significantly decreased contraction and relaxation under basal and β-adrenergic stress compared to nontransgenic hearts, with a reduction in maximal Ca2+-dependent force and maximal Ca2+-activated Mg2+-ATPase activity. However, Ca2+ sensitivity of force development and cTnI-Ser23/24 phosphorylation were not affected. Chemical shift mapping shows that both cTnI and cTnI{Delta}2–11 interact with the N lobe of cardiac troponin C (cTnC) and that phosphorylation at Ser23/24 weakens these interactions. These observations suggest that residues 2–11 of cTnI, comprising the acidic N' region, do not play a direct role in the calcium-induced transition in the cardiac regulatory or N lobe of cTnC. We hypothesized that phosphorylation at Ser23/24 induces a large conformational change positioning the conserved acidic N region to compete with actin for the inhibitory region of cTnI. Consistent with this hypothesis, deletion of the conserved acidic N' region results in a decrease in myocardial contractility in the cTnI{Delta}2–11 mice demonstrating the importance of acidic N' region in regulating myocardial contractility and mediating the response of the heart to β-AR stimulation.—Sadayappan, S., Finley, N., Howarth, J. W., Osinska, H., Klevitsky, R., Lorenz, J. N., Rosevear, P. R., Robbins, J. Role of the acidic N' region of cardiac troponin I in regulating myocardial function.


Key Words: muscle • transgenic mouse model • contractility • heart


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
β-ADRENERGIC (β-AR) SIGNALING PLAYS a fundamental role in modulating cardiac performance (1) . Physiological effects include increases in contractile force, heart rate, and the rate of relaxation. The use of β-AR agonists and β-AR blockers to treat acute ventricular failure or chronic failure, respectively, likely represent a delicate balance of different cardioprotective mechanisms. During β-AR stimulation, multiple proteins in the sarcolemma, sarcoplasmic reticulum, and myofilament are phosphorylated at multiple sites. One of the sarcomeric proteins, Cardiac troponin I (cTnI), is a key regulatory protein of the thin filament. There are three closely related troponin I (TnI) genes, each of which is selectively expressed in either the cardiac, fast skeletal, or slow skeletal muscle fibers. The embryonic heart expresses mostly slow skeletal muscle TnI, but expression gradually decreases during prenatal heart development as cTnI increases and becomes the only TnI isoform in the adult heart (2 3 4) . cTnI interacts with the major proteins present in the sarcomeric thin filament, including actin, cTnC, {alpha}-tropomyosin ({alpha}-TM), and troponin T (cTnT). These interactions underlie its central role as a molecular switch, regulating muscle contraction in response to changes in intracellular Ca2+ concentrations.

cTnI differs from the slow skeletal isoform of TnI in that it contains a 32 amino acid (31 in the human) N' extension. The N' extension of cTnI is composed of three regions; an acidic N' region containing a single turn of helix, an extended rigid polyproline helix, and a C' helix containing the bisphosphorylation motif (5) . The β-AR signaling pathway controls phosphorylation of the two serine residues (Ser-23/24) in the N' extension by cAMP-dependent protein kinase (PKA; ref. 6 ) and protein kinase D (PKD) (7) . Phosphorylation of Ser-23/24 results in a reduction in myofilament Ca2+ sensitivity (1) and an increase in cross-bridge cycling rate (8) by reducing the Ca2+-binding affinity of cTnC and allowing fine tuning of contractile function (9) . This mechanism plays an important role in the functional adaptation of cardiac muscle to physiological or pathological stress.

Atomic resolution structures, uniform density models of troponin derived from neutron contrast variation data, and molecular modeling data have enabled predictions to be made and tested for functional domains of cTnI. In the nonphosphorylated state, the N' extension interacts with cTnC’s inactive Ca2+-binding site I and helix A, largely through a series of weak electrostatic and hydrophobic interactions such that the acidic N' region does not strongly contact cTnC (10 11 12) . However, an important unanswered question is the role of the conserved acidic N' region of cTnI in modulating cardiac function. Based on the available biochemical and physiological data, we recently tested the hypothesis that modulation through phosphorylation might be partially mediated by electrostatic interactions between cTnI’s acidic N' region and available basic regions in cTnI, altering cross-bridge kinetics (5) . Thus, the role of phosphorylation would be to stabilize a conformation able to facilitate these ionic interactions. Phosphorylation-induced loss of interaction with the N' lobe of cTnC probably induces a hinge movement of the cardiac-specific N' extension, positioning cTnI’s acidic N' region for electrostatic interaction with the conserved basic region of cTnI (5) . The poly(L-proline) II (PPII) helix in the cardiac N' extension serves as a rigid spacer to position the acidic N' region near basic regions in cTnI. PPII helices are well suited for such conformational movement due to their restricted conformational rigidity, solvent exposure, and ability to present a hydrophobic surface as well as hydrogen bonding sites. Consistent with this hypothesis, nuclear magnetic resonance (NMR) and modeling studies showed that bisphosphorylation of cTnI at Ser-23/24 resulted in bending of cTnI and positioning of the acidic N' region for interaction with the basic inhibitory region of cTnI (5) .

To determine the role of the acidic N' region of cTnI on cardiac contractile function, we generated transgenic (TG) mice with cardiomyocyte-specific postnatal overexpression of a truncated cTnI that lacks the acidic N' region (cTnI{Delta}2–11). cTnI{Delta}2–11 cardiac myofibrils showed reductions in both maximal Mg2+-ATPase activity and absolute force but no changes in Ca2+ affinity. cTnI{Delta}2–11 hearts had significantly reduced rates of contraction and relaxation under baseline and β-agonist treatment. These findings indicate that the acidic N' region of cTnI plays an important role in regulating cardiac function in nonstimulated hearts as well as during β-AR stimulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of TG mice
To investigate the role of the acidic N' region of cTnI on cardiac contractile function, we generated a cDNA that encodes mouse cTnI with a deletion of residues 2–11 (ADESSDAAGE). The full-length mouse wild-type (WT) cDNA cTnI was obtained by reverse transcription-polymerase chain reaction (PCR) using total RNA isolated from mouse cardiac ventricles. The cDNA containing cTnIWT fragments were initially subcloned into pBluescript and sequenced as described earlier (13) . The 10 amino acids of the acidic N' region (Fig. 1 A) were deleted by standard PCR-based methods (cTnI{Delta}2–11). The cTnI{Delta}2–11 cDNA was subcloned into a site immediately downstream of the mouse cardiac {alpha}-myosin heavy chain promoter ({alpha}-MyHC), and the sequence was verified by DNA sequencing. The TG cassette was then released from the vector backbone using NotI digestion, followed by gel purification. Multiple lines of FVB/N TG mice were generated using the purified NotI-digested DNAs. Founder mice were identified by PCR using tail clip DNA as template. Transgene copy number was determined by Southern blot analysis using an {alpha}-MyHC promoter probe. The founders were bred to nontransgenic (NTG) mice and lines showing Mendelian patterns of transmission selected for further analysis. Five or six mice per experiment, 12- to 15-wk-old of mixed gender were used for our studies after pilot experiments showed no gender differences. All TG mouse lines were viable and fertile. The mice had a normal life span and no gross cardiovascular pathology presented. All protocols complied with the Guide for the Use and Care of Laboratory Animals published by the U.S. National Institutes of Health.


Figure 1
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  		Figure 1. Cardiac specific N' extension and RNA expression in cTnI{Delta}2–11 mice. A) Conserved N' extension in cTnI. *Amino acid residues identical to the human cTnI. The N' extension, residues 1–31, is marked with arrows. The acidic N' region, which was deleted in the cTnI{Delta}2–11 hearts, and the phosphorylation motif are each shaded, and the polyproline (Xaa-Pro) spacer arm is indicated. B) RNA dot-blot analyses of cardiac gene expression in 12-wk-old cTnI{Delta}2–11 TG lines (93, 81, and 78) compared to a NTG and cTnIWT TG line (line 52; James et al., ref. 13 ). C) Fold changes in the amount of cTnI transcript levels normalized to GAPDH. Values are mean ± SE (n=4). *P < 0.001.

Molecular and protein analyses
Transcript levels were determined by RNA dot blot analysis with {gamma}-32P-labeled cTnI and human growth hormone as well as probes for the cardiac hypertrophic markers atrial natriuretic factor (ANF) and β-MyHC (14) . Myofibrillar proteins were isolated from NTG and cTnI{Delta}2–11 mouse hearts using F60 buffer as described previously (9) and assayed for protein concentrations using the Bradford method (Bio-Rad, Hercules, CA, USA). The percentage of cTnI replacement was determined via SDS-PAGE (4–15% gradient Tris-HCl Ready Gel; Bio-Rad) and Western blots using polyclonal antibodies against cTnI (Cell Signaling Technology, Danvers, MA, USA). Two-dimensional gel electrophoresis was carried out as described previously (9) . Antibodies used for Western blot analysis are as follows: phospho-specific cTnI-Ser23/24 (Cell Signaling Technology), total phospholamban (PLN; Upstate, Lake Placid, NY, USA), phosphorylated PLN-Ser16 (Upstate), phosphorylated PLN-Thr17 (Cyclacel, Dundee, UK), calsequestrin (Research Diagnostics, Flanders, NJ, USA), cardiac myosin binding protein-C (cMyBP-C) C0-C1 domain (14) , phospho-specific cMyBP-CSer-282 (generous gift of Lucie Carrier, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; ref. 15 ), cardiac troponin T (Sigma, St. Louis, MO, USA), and cardiac {alpha}-tropomyosin (Chemicon, Temecula, CA, USA).

Histopathology and immunohistochemistry analyses
The heart weight (HW) and the ratio of heart weight:body weight (HW/BW) were measured to determine if cardiac hypertrophy had occurred. Gross examination and histopathological analysis were carried out as described previously (9) . The paraffin-embedded longitudinal sections of whole mouse hearts stained with hematoxylin-eosin or Masson’s trichrome were examined for overall morphology, presence of necrosis, fibrosis, myocyte disarray, and calcification using an Olympus B-60 microscope and SPOT software (Diagnostic Instruments, Sterling Heights, MI, USA). Localization and integration of cTnI{Delta}2–11 into the sarcomere were determined by confocal microscopy (14) . Five-micrometer cryostat sections were probed with cTnI antibodies (Cell Signaling Technology) followed by incubation with Alexa-488 conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA).

Cardiac function and β-AR responsiveness
For two-dimensional M-mode echocardiography, mice with the implanted osmotic pumps were anesthetized with 2% isoflurane. Hearts were visualized with a Hewlett-Packard (Palo Alto, CA, USA) Sonos 5500 instrument and a 15 MHz transducer (14) . Measurements were taken three times per mouse from different areas and then averaged for left ventricular (LV) diastolic and systolic dimensions and septal and posterior wall thickness, from which fractional shortening (FS) and LV mass was derived. Invasive hemodynamic studies were performed in the intact animals as described previously (16) . Data were analyzed using a PowerLab system (ADInstruments, Colorado Springs, CO, USA).

Chronic isoproterenol (ISO) infusion
To determine the stress tolerance of cTnI{Delta}2–11 hearts, NTG and cTnI{Delta}2–11 animals underwent 2 wk of continuous infusion of the β-agonist ISO (Sigma). Alzet miniosmotic pumps (Durect Corporation, Cupertino, CA, USA) containing either ISO (60 mg/kg/day) in 0.02% ascorbic acid (Sigma) or vehicle only (sham) were surgically implanted between the scapulae in 12-wk-old NTG and cTnI{Delta}2–11 mice for 14 days as described previously (17) . Cardiac function was measured by M-mode echocardiography before and 7 and 14 days after implantation.

In vitro PKA phosphorylation and Ca2+-activated Mg2+-ATPase assays
To phosphorylate myofibrillar proteins, total myofibrils were incubated with the catalytic subunit of PKA as described earlier (9) . Ca2+-activated Mg2+-ATPase activity was measured by titrating Ca2+ sensitivity of the NTG and cTnI{Delta}2–11 mouse hearts and measuring inorganic phosphate (Pi) release (9) . Data were analyzed by fitting the data obtained for each individual and then averaging the derived Hill parameters as described previously (9) .

In situ fiber kinetics
Procedures for mechanical analysis of murine papillary fibers have been described previously (18 , 19) . In brief, mice were injected with heparin (500 IU/kg intraperitonally) 5 min before being killed. To prepare skinned fibers, the heart was removed and placed in relaxing solution (5.37 mM ATP, 30 mM phosphocreatine, 5.0 mM EGTA, 20 mM BES, 7.33 mM MgCl2, 0.12 mM CaCl2, 10 mM DTE, 10 µg/ml leupeptin, and 32 mM potassium methansulfonate, pH 7.0) at 4°C. The solution also contained 30 mM of 2,3-butanedione monoxime designed to protect myocardial tissue from mechanical injury. Muscle fibers of ~0.5 mm diameter and 2–3 mm length were isolated from left ventricular papillary muscles. The fiber strips were skinned by incubation in 5.5 mM ATP, 5.0 mM EGTA, 20 mM BES, 6.13 mM MgCl2, 0.11 mM CaCl2, 10 mM DTE, 10 µg/ml leupeptin, 121.8 mM potassium methansulfonate (pH 7.0), and 50% glycerol with 0.5% (w/v) Triton X-100 for 12 h at 4°C. The fiber strips were then transferred to fresh solution without Triton X-100 and stored at –20°C until used. Dissected fibers were mounted isometrically between a force transducer and a length-step generator in relaxing solution (Scientific Instruments, Heidelberg, Germany). Sarcomere length (determined by laser diffraction analysis) at resting tension was always 2.0–2.1 µm. We determined that the cross-sectional area at the base of the muscle was between 0.05 to 0.1 mm2. Contraction solution had the same composition as the relaxing solution, except that EGTA was substituted with 5 mM [Ca2+]EGTA. Initial maximum isometric force was measured in activating solution (pCa 5.0). Force was determined and recorded on a chart recorder while the fibers were bathed in sequentially increasing Ca2+ concentrations ranging from pCa 8 to 5.0. Strip tension (mN/mm2) was calculated by dividing force by fiber cross-sectional area, calculated from widths measured at the major axis. To examine the effects of PKA phosphorylation on the pCa-force relationship in vitro, skinned fibers were treated with PKA (Novagen, San Diego, CA, USA). After measurements of the pCa-force relationship (before PKA treatment), the fiber was incubated in relaxing solution (pCa 8.0) plus 0.5 µM PKA for 10 min. The fibers were relaxed for 15 min, and the pCa-force relationship was measured after PKA treatment.

NMR spectroscopy and data processing
The cTnIWT and cTnI{Delta}2–11 cDNAs were subcloned into the pET23a+ expression vector (Novagen). [15N,2H]cTnC, cTnIWT, and cTnI{Delta}2–11 proteins were expressed in bacteria, purified, and complex formation carried out as described previously (20 , 21) . NMR experiments were performed at 40°C on Varian 600 or 800 MHz Inova spectrometers (22) . Spectral widths in the t1 and t2 dimensions were 3.3 and 12 kHz, respectively. Composite amide chemical shift differences were determined from the square root of the weighted sum of the squares of proton and nitrogen chemical shift differences. 15N chemical shift differences were weighted by a factor of one-seventh to scale their contributions to a magnitude similar to 1H chemical shift differences and the data processed as described previously (21) . Spectra were processed with Felix 2000 and analyzed with Sparky (T. D. Goddard and D. G. Kneller, Sparky3, University of California, San Francisco, CA, USA) software packages.

Statistical analysis
All values are expressed as means ± SE. The statistical significance of differences between two groups and multiple groups was determined by Student’s t test and two-way ANOVA (SigmaStat 3.1, Systat Software, San Jose, CA, USA), respectively. For all tests, P < 0.05 was considered significant. The Hill coefficient was calculated using Origin 7.5 NLSF tool (OriginLab Corporation, Northampton, MA, USA) as described previously (9) . The theoretical molecular weight and isoelectric point of cTnI were calculated at http://prometheus.brc.mcw.edu/promost/.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cardiac specific expression of the cTnI{Delta}2–11 transgene
To investigate the functional effects of cTnI’s acidic N' region (Fig. 1A ) in intact mouse hearts, we generated TG mice in which this region was deleted (cTnI{Delta}2–11). The cDNA was linked to the mouse {alpha}-MyHC promoter to drive cardiac-specific expression and used to generate multiple TG mouse lines. Nineteen TG lines were obtained with varying copy numbers as analyzed by Southern blot analysis. For all of the lines, normal Mendelian ratios were observed, indicating that expression did not result in any detectable embryonic lethality. The amount of TG transcript being expressed was determined for each line, and three lines (lines 93, 81, and 78) were selected in which cTnI{Delta}2–11 expression was roughly equivalent to a TG line that expresses high levels of normal cTnI (cTnIWT; Fig. 1B ), which served as a control to rule out any phenotype that might occur merely as a result of high TG expression levels of cTnI (13) . Lines 93, 81, and 78 showed 5.5-, 4.0-, and 2.8-fold increases over NTG controls, respectively (Fig. 1C ). The sarcomere has the capacity to maintain protein stoichiometry even when the transcript levels are increased via TG manipulation. Thus the degree of replacement is a function of the level of overexpression (9) . The degree of cTnI{Delta}2–11 protein replacement was confirmed by SDS-PAGE (Fig. 2 A) as well as by Western blots using cTnI-specific antibodies that recognize both the endogenous cTnI and TG cTnI{Delta}2–11 proteins (Fig. 2B ). Densitometry showed that we obtained essentially complete replacement of the endogenous protein with the transgenically-encoded cTnI{Delta}2–11 in line 93, which was chosen for subsequent functional analyses. Replacement of endogenous cTnI with cTnI{Delta}2–11 had no effect on expression levels of the other major contractile proteins, including MyHC, actin, myosin light chains, cTnT, and {alpha}-TM (Fig. 2A ). The deletion of the acidic N' region of cTnI appeared to be benign with no signs of increased morbidity, mortality, or cardiac hypertrophy, implying that cTnI{Delta}2–11 is not detrimental to gross cardiac morphology. HW/BW did not differ significantly between NTG (0.0051±0.006; n=8) and cTnI{Delta}2–11 (0.0050±0.005; n=6) littermates. The cTnI{Delta}2–11 hearts were unremarkable and showed no evidence of abnormal morphology, myofibrillar disarray, necrosis, or ventricular fibrosis (Fig. 3 A, B). Normal integration of cTnI{Delta}2–11 into the sarcomere was confirmed by immunofluorescent detection using cTnI-specific antibodies (Fig. 3C ).


Figure 2
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  		Figure 2. Myofilament protein composition, expression of, and replacement with cTnI{Delta}2–11. A) Total myofibrillar proteins from 3 TG lines generated with the cTnI{Delta}2–11 construct were analyzed to determine the degree of cTnI{Delta}2–11 replacement and conservation of the other sarcomeric protein levels. cTnI{Delta}2–11 migrates faster in SDS-PAGE relative to endogenous cTnI due to the deletion of the first 10 amino acids. The level of replacement can be clearly seen. A region of the gel containing cTnI, cTnI{Delta}2–11, and ELC1v was scanned, and the relative intensities are shown. B) Western analyses shows that cTnI{Delta}2–11 expression did not alter the total cTnI protein content. A representative histogram of cTnI and cTnI{Delta}2–11 is shown (n=3). {alpha}-sarcomeric actin was used as a loading control.


Figure 3
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  		Figure 3. Histopathological analyses. A, B) Longitudinal sections derived from 3-month-old left ventricle stained with hematoxylin-eosin (A) or Masson trichrome (B) demonstrate the lack of obvious pathology (x20). C) Immunofluorescent staining of cTnI with anticTnI polyclonal antibodies shows normal incorporation into the sarcomere (x60).

Functional analyses of cTnI{Delta}2–11 hearts
Although the TG animals appeared overtly normal and showed no obvious pathology, we hypothesized that the cTnI{Delta}2–11 hearts would have reduced basal cardiac function and this deficit would remain or become more pronounced during β-AR stimulation. To test this, NTG and cTnI{Delta}2–11 animals underwent 2 wk of continuous β-agonist infusion with ISO. We utilized four, 12-wk-old groups; NTG (sham), NTG (ISO), cTnI{Delta}2–11 (sham), and cTnI{Delta}2–11 (ISO). At the end of the 2 wk infusion, cardiac function was assessed by M-mode echocardiography. Both the NTG and cTnI{Delta}2–11 sham animals showed normal fractional shortening, and the other functional parameters did not differ between the two groups (Fig. 4 A, C; Table 1 ). In contrast, both the NTG and cTnI{Delta}2–11 ISO groups had significantly increased left ventricular inner diameters in diastole and systole and decreased fractional shortening. Importantly, significant differences presented between the NTG and cTnI{Delta}2–11 groups, with the cTnI{Delta}2–11 ISO hearts displaying both decreased heart rates and fractional shortening compared to the NTG ISO hearts. Continuous ISO infusion increased the HW/BW significantly in both NTG and cTnI{Delta}2–11 ISO groups compared to the respective sham groups, but no differences presented between the NTG and cTnI{Delta}2–11 ISO groups, ruling out a differential hypertrophic response as being responsible for the functional differences that were observed (Fig. 4B, D ).


Figure 4
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  		Figure 4. In vivo cardiac function. Alzet osmotic minipumps (model 2002; Alza) that contained either ISO (60 mg/kg/day) or PBS (sham) were implanted dorsally and subcutaneously under isoflurane anesthesia. In vivo cardiac function was evaluated in the following groups; NTG/sham, NTG/ISO, cTnI{Delta}2–11/sham, and cTnI{Delta}2–11/ISO. Mice were 12 wk of age and were implanted with the pumps for a total of 14 days of stimulation. A) The fractional shortening (%) was measured by M-mode echocardiography. *Significant difference vs. NTG (P<0.01; n=6). ##Significant difference vs. NTG-ISO (P<0.01, n=6). B) HW/BW for adult mice. **Significant difference vs. NTG (P<0.01; n=6). C) M-mode echocardiographic tracings show LV dilation in the NTG/ISO and cTnI{Delta}2–11/ISO hearts. D) Longitudinal sections stained with hematoxylin/eosin (x4) show enlarged LV in the NTG/ISO and cTnI{Delta}2–11/ISO hearts. E) Maximal rate of ventricular contraction (dP/dtmax). F) Maximal rate of ventricular relaxation (dP/dtmin). Data were analyzed using a mixed, two-factor analysis of variance with repeated measures on the second factor. **P < 0.01; *P < 0.001, significant difference vs. NTG (n=6). All data are presented as mean ± SE.


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  		Table 1. Cardiac function assessed by M-mode echocardiography

After the 2 wk infusion, in vivo hemodynamics were determined using the closed chest model and invasive catheterization (see Materials and Methods). Ventricular contraction and relaxation were measured at baseline and during dobutamine infusion. As expected, dobutamine infusion increased dP/dtmax and dP/dtmin in both the NTG and cTnI{Delta}2–11 sham hearts (Fig. 4E, F ) but basal and stimulated values for both these parameters were decreased in the cTnI{Delta}2–11 hearts as compared to the NTG hearts. That is, dP/dtmax increased in NTG sham hearts by 50% at the highest dobutamine dose, but only by 30% in the cTnI{Delta}2–11 sham hearts (Fig. 4E, F ; Table 2 ). After 2 wk of continuous ISO infusion, the cTnI{Delta}2–11 ISO hearts displayed a relatively depressed heart rate at baseline and on dobutamine infusion (Table 2) . Hemodynamic parameters, including LV pressure, dP/dtmax, and dP/dtmin, were also significantly compromised in the cTnI{Delta}2–11 ISO hearts, compared to the NTG ISO group (Table 2) . The NTG ISO hearts showed conserved cardiac function with values for both dP/dtmax and dP/dtmin at baseline almost equivalent to the rates that obtained with a maximum dose of dobutamine in the NTG sham mice. As expected, NTG ISO hearts were unable to respond to increasing concentrations of dobutamine as they were already fully stimulated. These results indicate that, despite appearing overtly normal and healthy under standard cage conditions, the acidic N' region deletion does compromise cardiac function and this is exacerbated under conditions of chronic and acute β-AR stimulation.


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  		Table 2. In vivo hemodynamic measurements

Actomyosin Ca2+-activated maximal Mg2+-ATPase and Ca2+ sensitivity
We used skinned papillary muscle fibers to assess the effect of cTnI{Delta}2–11 on the kinetics and Ca2+ sensitivity of force generating cross-bridge formation. Fiber preparations were also subjected to PKA treatment to compare fully phosphorylated cTnI with the truncated TG protein (Fig. 5 A). Average maximally developed isometric tension (Fmax) was significantly decreased in cTnI{Delta}2–11derived fibers relative to NTG fibers (8.90±0.4 vs. 5.99±0.6 kN/m2; P<0.002; n=5), although Ca2+ sensitivity was unaffected. The same fibers were then treated with PKA, and the effects on Ca2+ sensitivity and maximum force were determined for both the NTG and cTnI{Delta}2–11 fibers. Exogenous treatment of fibers with PKA did not affect Fmax in either group (Table 3 ). Ca2+ sensitivity was significantly decreased by PKA treatment compared to the untreated fibers for both groups. In this isolated system, the effect of cTnI{Delta}2–11 on maximal force development is consistent with decreased myocardial contractility in vivo and a concomitant decrease in the number of force-generating cross-bridges. There was no difference in the basal and PKA-activated Ca2+-sensitivity, indicating that the deletion did not affect phosphorylation of Ser-23/24 or myofilament Ca2+ sensitivity.


Figure 5
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  		Figure 5. In vitro skinned fiber analyses. A) Force-pCa relationship of chemically skinned ventricular fibers before and after PKA-mediated phosphorylation. A bundle of 3–5 fibers isolated from glycerinated mouse papillary muscle fibers was attached by tweezer clips to a force transducer. After the initial steady-state force was measured in pCa 5.0 solution, the fiber bundles were exposed to increasing Ca2+ concentrations (pCa 8.0 to 5.0). After a contraction-relaxation cycle in the absence of PKA, the fibers were incubated with 0.5 µM PKA for 15 min at room temperature in relaxation solution (pCa 8.0). A second contraction-relaxation cycle was subsequently obtained under the same conditions. Force is expressed as % of the force obtained at maximal Ca2+ activation (pCa 5.0). Values are mean ± SE (n=5 per group; Table 3 ). B) Maximum Ca2+-dependent Mg2+-ATPase activity was determined at pCa 4.0. Activity measured at pCa 8.0 was subtracted and the activity at each pCa value was normalized to the maximum at pCa 4.0 for each experiment. Data points represent the mean ± SE of 5 separate experiments in triplicate (Table 4) . cTnI{Delta}2–11 myofibrils showed significantly reduced maximal Mg2+-ATPase activity under baseline and PKA treatment compared to NTG. In contrast, there were no differences in the Ca2+ sensitivity between NTG and cTnI{Delta}2–11 myofibrils. Inset: Representative immunoblot shows cTnI phosphorylation at Ser-23/24 by PKA (top). The blot shows protein derived from NTG and cTnI{Delta}2–11 (TG) hearts, untreated or treated with PKA, with phospho-cTnI antibody used as probe. {alpha}-Sarcomeric actin staining confirms equal loading (bottom).


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  		Table 3. Ca2+-activated maximum developed force

To understand the consequences of cTnI{Delta}2–11 on the Ca2+-activated actomyosin Mg2+-ATPase activity, myofibril Mg2+-ATPase activity and Ca2+ sensitivity were measured with or without PKA treatment (Fig. 5B ; Table 4 ). The cTnI{Delta}2–11 myofibrils showed a significant decrease in the maximal Mg2+-ATPase activity (163.07±4.85 nmol Pi/min/mg, P<0.01) compared to the NTG (188.96±4.67 nmol Pi/min/mg), but Ca2+ sensitivity was unaffected. As expected, PKA treatment of either cTnI{Delta}2–11 or NTG myofibrils resulted in decreased Ca2+ sensitivity. Hill coefficients and EC50 values fall within the range of those previously reported (9) . The Hill coefficients for the NTG and cTnI{Delta}2–11 proteins did not differ, reflecting the similarities in the shape of the Ca2+ binding curves. Phosphorylation of cTnI residues was confirmed by Western blots using phospho-specific cTnI antibodies (Fig. 5B , inset). Taken together, the data show that the acidic N' region plays a major role in regulating the actomyosin Ca2+-activated maximal Mg2+-ATPase activity.


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  		Table 4. Ca2+-activated Mg2+-ATPase activity

To confirm that these functional, hemodynamic, and biochemical changes were not due to compensatory changes in contractile protein phosphorylation states, PLN (Fig. 6 A), the myosin light chains (Fig. 6B ), and cMyBP-C (Fig. 6C ) phosphorylation was examined by Western blot analysis using phospho-specific antibodies and two-dimensional electrophoresis. The data showed that phosphorylation levels of these proteins were unchanged between the cTnI{Delta}2–11 and NTG sham and ISO hearts, respectively.


Figure 6
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  		Figure 6. Phosphorylation of PLN and myosin light chains. Cardiac total homogenates (10 µg) were subjected to 4–15% SDS-PAGE, electroblotted onto nitrocellulose membranes and probed with the indicated antibodies. A) Analyses of total PLN (PLN-Total), phospho-Ser-16 (PLN-Ser-16), and phospho-Thr-17 (PLN-Thr-17). PLNM and PLNP indicate monomeric and pentameric forms of PLN, respectively. Calsequestrin (Calse) was used as a loading control. B) Representative SYPRO Ruby stained 2D SDS-PAGE showing the phosphorylated species (arrowheads) of the essential light chain 1v (ELC1v) and regulatory light chain 2v (RLC2v). Total myofibrillar protein (75 µg) from ventricles was separated in the pH range of 4–7. C) cMyBP-C phosphorylation states in the NTG and TG groups were compared by Western blot analysis with 5 µg of protein isolated from 12-wk-old mouse hearts. Antibodies against cMyBP-C and phospho-cMyBP-C (pSer282) were used. Protein loading was normalized using total cTnT and cadiac {alpha}-TM antibodies. PLN, myosin light chains, and cMyBP-C phosphorylation levels did not differ between the NTG and cTnI{Delta}2–11 (TG) groups at baseline or between the groups after ISO treatment.

Structural and computational analysis of cTnI{Delta}2–11
Backbone amide resonances provide excellent probes for monitoring protein-protein interactions and identifying interaction surfaces. Chemical shift differences can be used to map the cTnC binding site of the N' extension (residues 2–33) of cTnI on Ca2+-loaded cTnC. Interactions of the N' extension (residues 2–33) and, in particular, the conserved acidic N' region (residues 2–11) were analyzed by comparing combined amide 1H and 15N chemical shift differences for Ca2+-loaded cTnC bound to cTnI and cTnI{Delta}2–33 (Fig. 7 A) and for cTnC bound to cTnI and cTnI{Delta}2–11 (Fig. 7B ). Residues showing significant chemical shift perturbations induced by the N' extension (residues 2–33) were located in the N' lobe of cTnC with the largest perturbations centered in defunct Ca2+-binding site I (residues 28–38) and Ca2+ binding site II (residues 65–76) (Fig. 1A ). Bis-phosphorylation or introduction of negative charge at Ser-23/24 in the N' extension results in a weakening of these interactions (11 , 12 , 23) . No significant chemical shift perturbations were observed in the linker region or in the C-lobe of cTnC. Composite amide 1H and 15N chemical shift differences for Ca2+-loaded cTnC bound to cTnI and cTnI{Delta}2–11 were considerably smaller, generally <0.05 ppm (Fig. 7B ). The magnitude of the observed chemical shift perturbations suggests that residues 2–11, comprising the conserved acidic N' region, do not interact strongly with Ca2+-loaded cTnC. This is consistent with previously published chemical shift perturbation analysis (23 , 24) and binding studies (25) supporting interactions between residues 19–30 in the N' extension and the N' lobe of Ca2+-loaded cTnC.


Figure 7
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  		Figure 7. Effect of cTnI{Delta}2–11 on N' lobe conformational states in cTnC. A, B) Combined amide proton and amide nitrogen absolute value chemical shift differences between Ca2+-loaded [15N, 2H]cTnC bound to cTnI{Delta}2–33 (A) and to cTnI1–211 and Ca2+-loaded [15N, 2H]cTnC bound to cTnI{Delta}2–11 and to cTnI1–211 (B). The horizontal bar represents the average chemical shift difference plus 1 SD. The amide proton and amide nitrogen chemical shifts of resonances in the N' lobe of cTnC can be used to monitor the regulatory domain conformational states on binding cTnI. C) Overlapping segments of 15N-1H correlation spectra for Glu66 and Ile128 in Ca2+-loaded [15N, 2H]cTnC. Chemical shift changes in Glu66 monitor conformational substates reflecting structural transitions from more "closed" to more "open" N' lobe conformations of cTnC. Amide 1H-15N correlations for free Ca2+-loaded [15N, 2H]cTnC are shown in green. Amide 1H-15N correlations for Ca2+-loaded [15N, 2H]cTnC bound to cTnI{Delta}2–11 and cTnI1–211 are shown in red and blue, respectively.

Amide proton and nitrogen chemical shifts of residues in the N' lobe of cTnC can be used to monitor conformational states within the individual lobes of cTnC on binding cTnI (23 , 24) . The conformational transitions for two residues, Glu66 and Ile128, located in the N' and C' lobes of cTnC respectively, were examined in detail. Glu66 can exhibit conformational-dependent chemical shift changes that are correlated with open and closed N' lobe conformations (24) . The binding of full-length cTnI to Ca2+-loaded cTnC results in a downfield shift for the amide cross-peak of Glu66 (Fig. 7C ). This shift is consistent with an opening of the N' lobe of cTnC and exposure of the hydrophobic cleft for binding the switch region of cTnI (24) . A similar downfield shift for the amide cross-peak of Glu66 is observed in the presence of cTnI{Delta}2–11 (Fig. 7C ). However, the magnitude of the shift is intermediate between that observed for free Ca2+-loaded cTnC and the Ca2+-loaded cTnC/cTnI complex (Fig. 7C ). The magnetically different amide 1H-15N environments presumably result from alterations in the N' lobe conformational substates reflecting open/closed equilibria (24) , indicating that the conserved acidic N' region (residues 2 3 4 5 6 7 8 9 10 11 ) shows some propensity for modulating N' lobe conformational states. Loss of the conserved acidic N' region may alter the interaction of residues 19–30 of cTnI with the N' lobe of cTnC. It is known that phosphorylation of Ser-23/24 of cTnI results in weakening of the interactions between the N' extension and the N' lobe of cTnC, providing a molecular basis by which N' lobe conformational equilibria are modified in response to physiological stimuli (24) . No significant chemical shift changes in Ile128 were observed between Ca2+-loaded cTnC and Ca2+-loaded cTnC bound to either cTnI or cTnI{Delta}2–11 (Fig. 7C ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human cTnI has an unique N' extension that is not present in the fast and slow skeletal muscle isoforms. This extension contains a unique acidic N' region, a Xaa-Pro region (residues 12–18) and a bisphosphorylation motif, in which Ser-23/24 are substrates for PKA, PKD, and the Rho kinases. Bisphosphorylation at Ser-23/24 in the N' extension of cTnI, in response to β-adrenergic stimulation, modulates myofilament Ca2+ sensitivity and cross-bridge kinetics (6 , 8) . The extension is highly conserved among mammals, and its functional importance is underscored by the finding of two missense mutations within the N' extension that cause familial hypertrophic cardiomyopathy (FHC) in humans (16 , 17) . One mutation, FHCA2V (20) , is located in the acidic N' region and the second, FHCR21C, in the phosphorylation motif (21) . Although NMR and modeling studies have shown that the conserved acidic N' region of the N' extension can extend from its position on the N-lobe of cTnC and interact with the basic inhibitory region of cTnI, the role of the acidic N' region of cTnI in the modulation of cardiac contractility is not clear. A novel finding reported in the present study is the reduction of rate of contractile function at baseline and β-AR stimulation in cTnI{Delta}2–11 hearts lacking the acidic N-terminal region of cTnI. TG mice expressing cardiac-specific cTnI{Delta}2–11 were generated to determine the role of the acidic N' region of cTnI on myocardial function and the response of the heart to β-AR stimulation. Although cTnI{Delta}2–11 alters the conformational states of cTnI and results in decreased contractile function, replacement is not lethal (22 , 26) . cTnI{Delta}2–11 hearts, with > 95% of cTnI replaced by cTnI{Delta}2–11, appeared grossly normal. The cTnI{Delta}2–11 mice are benign without any obvious increased mortality or detectable cardiovascular pathology. The cTnI{Delta}2–11 mice were viable and fertile and exhibited normal ventricular weights and heart rates; however, significant differences in contractile function were evident. These data suggest that removal of acidic N' region of cTnI would not affect the heart adversely. Similar findings were observed in cardiac-specific TG mice that express either a lack of the N' extension, residues 2–28 of cTnI (22) , or replacement of cTnI with the slow skeletal TnI (26) , which differs from cTnI by the absence of unique 32 residues at the N' extension of cTnI. Conversely, the mice (13) and rabbits (27) expressing cTnI146Gly, a FHC mutation that is located within the inhibitory region, resulted in cardiomyocyte disarray and interstitial fibrosis and suffered premature death.

In the present study, the absolute force and Mg2+-ATPase activity data follow the same pattern in cTnI{Delta}2–11 myofilaments. The expected decrease in maximal force and actomyosin Mg2+-ATPase activity as a result of cTnI{Delta}2–11 was confirmed in the skinned fiber studies with no change in Ca2+ sensitivity of the cTnI{Delta}2–11. Our data further show that the Ser-23/24 phosphorylation and Ca2+ binding sites are unaltered by incorporation of cTnI{Delta}2–11. Thus, we think that the deletion alters the orientation of the N' extension of cTnI, which then affects the interaction between the acidic N' region and the inhibitory domain of cTnI with cTnC and actin. In addition, a basic C' region of cTnI, not observed in the cTnI crystal structure (28) , can interact electrostatically with actin (29) . Modeling studies suggest that Ca2+-induced translocation of cTnI’s mobile C-terminal domain to actin is triggered by the switch region’s binding to the N' lobe of cTnC. Thus, in the low Ca2+ state, both the inhibitory region and mobile C-terminal domain of cTnI bind to actin, forming an electrostatic clamp that pushes tropomyosin toward the outer domain of actin (29) . Our studies indicate that the acidic N' region of cTnI plays an active role in modulating the interactions of the inhibitory region and the C-terminal mobile domain of cTnI with {alpha}-actin, which could influence the position of tropomyosin binding on actin. Protein-protein interactions within tropomyosin can influence the movement and position of tropomyosin on the actin surface (30) .

Ser-23/24 phosphorylation within the N' extension of cTnI results in a reduction in myofilament Ca2+ sensitivity, an increase in cross-bridge cycling, and enhanced binding of cTnI to the thin filament (8 , 9 , 31 , 32) . Studies in the intact heart have demonstrated a significant role of cTnI phosphorylation for both afterload dependence of ejection and relaxation (33) as well as force frequency modulation (31) . To determine the functional consequences of the acidic N' region on β-AR stimulation, we examined contraction and relaxation under baseline conditions and during β-AR stimulation. Incorporation of cTnI{Delta}2–11 into the sarcomere reduces baseline values for dP/dtmax and dP/dtmin, indicating a negative regulatory mechanism of cardiac function followed by decreased maximal force and Mg2+-ATPase activity. Differences in the dobutamine response of the TG and NTG mice showed that replacement with cTnI{Delta}2–11 affects the ability of the heart to respond to β-AR stimulation. The data confirm that the global changes that have occurred at the cardiomyocyte level as a result of the TG substitution with the transgenically encoded protein result in altered organ response as well. Moreover, during chronic long-term β-AR stimulation with ISO, contractile function was blunted in the cTnI{Delta}2–11 mouse hearts. We were able to exclude the possibility that alterations in either phosphorylation levels or expression levels of the cMyBP-C, myosin light chains, and PLN were compensating for a reduction in contractile function in the cTnI{Delta}2–11 hearts. Consistent with altered interactions of mutant cTnI with the cTnC and actin, TG mice that express a cTnI that lacks residues 2–28 (22) and the cTnI146Gly (13) showed enhanced contractility with impaired relaxation at the whole heart level.

NMR data and sequence analyses indicate a loosely structured N' extension with a propensity for a helical region surrounding the bisphosphorylation motif (residues 20–24), followed by a helical C-terminal region in residues 25–30 (5) . An extended PPII helix (residues 11–19) appears to serve as a rigid linker that aids in positioning the acidic N' region. In this conformation, the N' extension of cTnI interacts weakly with the N' lobe of cTnC, modulating myofilament Ca2+ sensitivity. NMR studies (10 , 12 , 23 , 24 , 34) , binding studies (35) , deletion mutagenesis (25) , and cross-linking studies (36) have defined residues 19–30 as the minimal region of the N' extension necessary for interacting with the N' lobe of cTnC. Residues surrounding inactive Ca2+ binding site I in the N' lobe of cTnC may comprise the interaction site with the N' extension of cTnI. NMR studies further showed that interaction of the cardiac N-extension alters N' lobe conformational equilibria in cTnC, presumably toward more active/open conformations that are capable of binding the switch region of cTnI (10 , 24 , 35) . Introduction of a negative charge at Ser-23/24 via phosphorylation stabilizes and extends the C-terminal helix (residues 21–30) of the cardiac N' extension, further weakening its interaction with the N' lobe of cTnC, leading to the repositioning of the acidic N' region. This results in alterations in N' lobe conformational equilibria, presumably toward more closed states, resulting in decreased Ca2+ sensitivity. This mechanism utilizes the unique isoform differences in both cTnC and cTnI, providing a molecular switch for modulating Ca2+ sensitivity of cardiac muscle contraction.

Recently, the determined structure for the cardiac N-extension bisphosphorylated at Ser-23/24 was found to contain a C-terminal helix (residues 21–30) containing the phosphorylation motif, an extended PPII helix (residues 11–19), and an acidic N terminus with some propensity for helical structure (5) . With the use of this structure, the X-ray crystal structure of the cTnI core, and uniform density models of the cTnI subunits derived from neutron contrast variation data, atomic models were built that show the conformational transition induced by PKA phosphorylation at Ser23/24 of cTnI and suggest a molecular linkage between the cardiac N' extension and the inhibitory region of cTnI (5) . The extended PPII helix provides a rigid linker, extending across the N-lobe of cTnC, which aids in positioning the acidic N' region with basic regions in the troponin complex. This may induce a bending in the rod-like cTnI at the end that interacts with the cTnC/cTnT component (5) . Those data (5) and the data in Fig. 7 are consistent with the transition between the open and closed states depicted in the model (Fig. 8 ) that we hypothesize occur as a result of acidic N' region interaction with the basic residues in its inhibitory region, competing with actin for the inhibitory region of cTnI, or with the second actin-binding region of cTnI (Fig. 8) , resulting in the alteration of cross-bridge kinetics (5) .


Figure 8
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  		Figure 8. Model of cTnI and effects of the cTnI{Delta}2–11 deletion. Helices of cTnI and cTnT found in the core crystal structure of cardiac troponin are shown in yellow and orange, respectively (Takeda et al., ref. 28 ). The inhibitory region of cTnI is shown in green. The PPII helix (Xaa-Pro) and the C-terminal helix (Phos), residues 12–32, containing the bisphosphorylation motif in the N' extension of cTnI are shown in blue. The acidic N' region (N'), residues 2–11, of the N' extension is shown in black. Cardiac TnC is shown in red. A) In the nonphosphorylated complex, the N' extension of cTnI contacts the N' lobe of cTnC. B) Bisphosphorylation at Ser-23/24 in the N' extension induces a bending in the cTnI subunit, positioning the acidic N' region for electrostatic interactions with the inhibitory region of cTnI. C) Deletion of acidic N' region, residues 2–11, in bisphsosphorylated cTnI results in the loss of interactions between the N' extension and the N' lobe of cTnC, as well as the loss of interactions with the inhibitory region of cTnI.

Our data are consistent with the hypothesis that removal of the acidic N' region from cTnI prevents interaction of this region with the inhibitory domain of cTnI, permitting it to associate more readily with actin. As a result, enhancing the contact between actin and the inhibitory domain of cTnI might ultimately diminish cardiac contractility and maximal force of contraction. In support of our proposed model, the cTnI{Delta}2–11 myofilaments showed decreased maximal absolute force and Mg2+-ATPase activity, leading to reduction of contractile function at baseline and during β-AR stimulation. Taken together, the data indicate that the interaction of the acidic N' region with the inhibitory region of cTnI provides a novel mechanism by which the acidic N' region modulates cross-bridge kinetics and regulates actomyosin interactions. Additional biochemical and structural studies are warranted to gain insight into the molecular function of the acidic N' region and its roles in cardiac muscle acidosis and regulation of contraction.


   ACKNOWLEDGMENTS
 
This research was supported by the U.S. National Institutes of Health (grants HL-69799, HL-60546, HL-52318, HL-60546, and HL-56370 to J.R. and HL-83334 to P.R.R.); by the American Heart Association, Ohio Valley Affiliate (to S.S.); and by the U.S. Department of Defense (grant ARO MURI DAAD 19–02-1–0027 to P.R.R.).

Received for publication July 24, 2007. Accepted for publication September 27, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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